E-selectin-dependent cell adhesion antagonists

Abstract

The present invention provides antagonists to cell adhesion useful in controlling the negative effects of inflammation, and the metastasis of cancer cells. These antagonists are ligands to E-selectin containing the sialyl Le x structure, including sialyl Le x glycoproteins, sialyl Le x glycolipids, and sialyl Le x oligsaccharides, and other related sialyl Le x -containing molecules capable of inhibiting E-selectin mediated cell adhesion to endothelial cells. The present invention also provides antibodies against sialyl Le x determinants capable of interrupting E-selectin mediated cell adhesion, which are also considered antagonists according to the present invention. The present invention also provides methods of using the antagonists of the present invention to reduce inflammation, and methods to inhibit the process of metastasis by carcinogenic cells. The present invention-also provides nucleic acid molecules encoding the glycoprotein antagonists of the present invention, in particular soluble chimeric leukosialin, and vectors capable of expressing these nucleic acid molecules, as well as cells capable of producing sialyl Le x positive recombinant glycoproteins. The present invention further provides a method of determining metastatic potential by comparing the efficiency of E-selectin-mediated adhesion of cell samples. In addition the present invention provides a method of producing a preferred antagonist of the present invention, sialyl Le x positive glycoproteins, in particular, sialy Le x positive chimeric leukosialin.

We claim:  
     
         1 . An antagonist to E-selectin mediated cell adhesion wherein said antagonist is selected from a group of molecules consisting of a ligand to E-selectin containing sialyl Le x -termini, and an anti-sialyl Le x  antibody.  
     
     
         2 . The antagonist of  claim 1  wherein said ligand is selected from a group of molecules consisting of sialyl Le x -glycoproteins, sialyl Le x -glycolipids, and sialyl Le x -chimeric glycoproteins.  
     
     
         3 . The antagonist of  claim 1  wherein said sialyl Le x -containing chimeric glycoprotein is soluble chimeric leukosialin protein.  
     
     
         4 . The antagonist of  claim 3 , wherein said soluble chimeric leukosialin is chimeric soluble human leukosialin-IgG protein.  
     
     
         5 . A composition containing an effective amount of one or more of the antagonists of  claim 1  in addition to a carrier.  
     
     
         6 . A nucleic acid sequence encoding a soluble chimeric glycoprotein.  
     
     
         7 . A nucleic acid sequence encoding a soluble chimeric leukosialin-IgG.  
     
     
         8 . A vector containing the nucleic acid sequence of  claim 6 .  
     
     
         9 . A mammalian cell containing both a vector capable of directing the expression of a soluble glycoprotein and a vector capable of directing the expression of a fucosyltransferase enzyme which directs sialyl Le x  synthesis.  
     
     
         10 . A mammalian cell containing both a vector capable of directing the expression of a soluble chimeric leukosialin and a vector capable of directing the expression of a fucosyltransferase enzyme which directs sialy Le x  synthesis.  
     
     
         11 . The mammalian cell of  claim 9  wherein said cell is a CHO cell.  
     
     
         12 . A method of recombinantly producing glycoproteins expressing sialyl Le x  termini comprising transfecting a culture of appropriate mammalian cells with both a vector which directs the expression of a soluble glycoprotein and a vector which directs the expression of a fucosyltransferase enzyme.  
     
     
         13 . The method of  claim 12  wherein the glycoprotein produced is soluble sialyl Le x  containing leukosialin.  
     
     
         14 . The method of  claim 12  wherein the mammalian cells are CHO cells.  
     
     
         15 . A method of inhibiting E-selectin mediated adhesion of carcinoma cells to endothelial cells comprising administering the antagonist of  claim 1  to a subject.  
     
     
         16 . A method of inhibiting E-selectin mediated adhesion of carcinoma cells to endothelial cells comprising administering a sialyl Le x -glycoprotein, a sialyl Le x -glycolipid, or a sialyl Le x -chimeric glycoprotein to a subject.  
     
     
         17 . A method of determining the metastatic potential for a sample of carcinoma cells comprising determining the percentage of carcinoma cells which bind to E-selectin expressing cells.  
     
     
         18 . The method of  claim 17 , wherein said E-selectin expressing cells is selected from the group consisting of activated human endothelial cells, activated mouse endothelial cells, and CHO cells expressing E-selectin.  
     
     
         19 . A method of determining the metastatic potential for sample of carcinoma cells comprising determining whether an antagonist of  claim 1  inhibits in a concentration dependent manner the adhesion of the carcinoma cell sample to E-selectin expressing cells.  
     
     
         20 . The method of  claim 19 , wherein the E-selectin expressing cells are selected from the group consisting of activated human endothelial cells, activated mouse endothelial cells, and CHO cells expressing E-selectin.